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Mandlik et al. BMC Evolutionary Biology 2014, 14:142http://www.biomedcentral.com/1471-2148/14/142

RESEARCH ARTICLE Open Access

Molecular evolution of the enzymes involved inthe sphingolipid metabolism of Leishmania:selection pressure in relation to functionaldivergence and conservationVineetha Mandlik, Sonali Shinde and Shailza Singh*

Abstract

Background: Selection pressure governs the relative mutability and the conservedness of a protein across theprotein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressurethat arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL(Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in thesphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivityof the parasite and have been classified as druggable targets in the parasite metabolome.

Results: The present work relates to the role of selection pressure deciding functional conservedness anddivergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The threeprotein families were largely under the influence of purifying selection and were moderately conserved baring tworesidues in the IPCS protein which were under the influence of positive selection. To further explore the selectionpressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymouscodon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families.

Conclusion: Evolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acidslining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 ofIPCS) should be avoided during devising strategies for inhibitor design.

Keywords: Evolutionary biology, Sphingolipid metabolism of Leishmania, Functional divergence and conservedness,Specificity determining positions, Selection pressure, Codon usage bias, Relative synonymous codon usage, Effectivenumber of Codon, GC content

BackgroundLeishmania, a protozoan parasite is responsible for caus-ing the infectious disease Leishmaniasis. Around 12 mil-lion people are affected by this disease worldwide. Thesphingolipid metabolism of vertebrates, fungi and plantshas been well documented. Sphingolipid metabolism inparasites like Leishmania plays an important role in main-taining the infectivity of the parasite. Sphingolipids form anintegral component of the parasitic membranes [1]. Theyare localized in the membrane micro domains and are

* Correspondence: [email protected] Centre for Cell Science, NCCS Complex, Pune University Campus,Ganeshkhind, Pune 411007, India

© 2014 Mandlik et al.; licensee BioMed CentraCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.

involved in a wide array of signal transduction pathways [2]Parasites like Leishmania scavenge host sphingolipids andremodel them into parasite specific sphingolipids. The keyenzymes involved in the sphingolipid metabolism are a)SPT (Serine pamitoyl transferase) b) SPL (Sphingosine 1-Pphosphate) and c) IPCS (Inositol phosphorylceramidesynthase) [3].SPT, the first key enzyme in the sphingolipid metabolism

localizes into the endoplasmic reticulum and belongs to theclass-II pyridoxal-phosphate-dependent aminotransferasefamily [4]. It comprises of two subunits SPTLC2 andSPTLC3 and catalyses the first step of the de novo synthesisof sphingolipids i.e. condensation reaction of serine and

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palmitoyl-CoA to form 3-dehydro D-sphinganine. The sec-ond key enzyme, SPL belongs to the Group II pyridoxal-dependent decarboxylase family of enzymes [5]. It acts onthe phosphorylated sphingoid bases (PSBs) such assphingosine-1-phosphate and cleaves them into aldehydesand phosphoethanolamine [6]. IPCS represents another keyenzyme in the sphingolipid metabolism. IPCS is unique tocertain fungi, plants and protozoan parasites like Leish-mania. There is no mammalian equivalent of this enzymeand thereby IPCS has been considered as an attractive drugtarget in the sphingolipid metabolism of Leishmania [7].IPCS localizes in the Golgi complex and catalyses the reac-tion involving the conversion of ceramide to inositol phos-phoryl ceramide (IPC) [8,9]. The importance and role ofthese three target proteins in the sphingolipid metabolismhas been shown in Figure 1.

Figure 1 Functional importance of the enzymes (IPCS, SPT and SPL) i

The sphingolipid metabolism of Leishmania and manyother pathogens is highly conserved and offers a series ofattractive drug targets for further inhibitor design. IPCS,SPT and SPL in Leishmania have been identified as im-portant target proteins by biochemical network modeling.[10] We present the phylogenetic relationship amongthese key enzymes in Leishmania to obtain a comparativehistory of the related proteins. Role of selection pressure,assessment of the strength of purifying versus diversifyingselection for all the three target proteins has provided anidea of the molecular evolution of target proteins.

MethodsAcquisition of sequencesAmino acid and coding sequences of the three enzymes(IPCS, SPL and SPT) in the sphingolipid metabolism were

n the sphingolipid metabolism of Leishmania.


retrieved from NCBI, ENSEMBLE and UNIPROT data-bases. A total of 54 sequences for IPCS and SMS, 40 se-quences of SPL and 78 sequences for SPT were used inthis study (Additional file 1: Table S1A-E).

Sequence alignment, selective constraints andphylogenetic analysisThe sequences were aligned using CLUSTALW v2.0.9using the default parameters. Larkin et al. [11] Phylogenetictree reconstruction for the sphingolipid metabolism wasdone using MEGA 5 program by the Neighbour-Joiningmethod with 10000 bootstrap resampling’s. Saitou and Nei

Figure 2 The phylogenetic tree depicting the evolutionary relationshsphingolipid metabolism in comparison to SMS enzyme in the host. Treplicates. Bootstrap values are indicated on the branches, depicting the ev

[12,13]. The evolutionary distances (Number of amino acidsubstitutions/site) were computed using the Poisson correc-tion method.

Functional divergenceTo evaluate the potential functional divergence and to pre-dict the amino acid residues accounting for the functionaldifferences among the three enzymes, Type I functionaldivergence was estimated using DIVERGE 2.0. Gaucheret al. [14] Sequences were classified into three differentgroups (IPCS, SPL and SPT) using the P-distancemethod (Additional file 1: Table S2). For each pairwise

ip of the three enzymes (IPCS, SPL and SPT) belonging to thehe tree was constructed by the NJ method using MEGA5 with 10000olutionary relationship among the three protein families.


comparison, the coefficient of evolutionary functionaldivergence (θ) and standard error were determined.Utilizing the coefficient of evolutionary functional diver-gence (θ) the sequences were subjected to significantfunctional divergence and likelihood ratio test (LRT).Based on the site-specific posterior probabilities, sitesexperiencing functional divergence in the subgroupswere identified [15,16].

SDP (Specificity determining positions) analysisSDP’s determine the differences in functional specifi-city within the protein family [17,18]. The input set ofsequences was divided into 4 groups (IPCS, SMS, SPLand SPT) containing 34, 20, 40 and 78 sequencesrespectively. SDP’S were predicted by SDPfox andSDPpred and the Z scores were calculated for eachalignment column.

Selection pressure assessment of the protein familiesThe selected clades from functional divergence weresubmitted as nucleotide alignment in fasta format to theSelecton server for analysis of the non-synonymous (dN)versus synonymous substitution (dS) ratio [19]. To studythe effect of selection pressure on the conservedness ofthe three proteins, the protein MSA was submitted tothe CONSURF server, which classifies the residues basedon their conservation in the MSA [20].

Selection pressure assessment at the codon levelSelection pressure on the codon was estimated for the tar-geted organism Leishmania. Protozoan coding sequences

Figure 3 Sites with altered evolutionary rates identified by posterior

were obtained from GenBank (release 137). In order tonormalize codon usage within datasets of differing aminoacid compositions, relative synonymous codon usage(RSCU) values were calculated. The reference set con-sisted of highly expressed genes pertaining to the lipid me-tabolism as reported by [21] Codon Adaptation Index(CAI) was calculated using CAI Calculator 2 [22]. Otherspecies non-specific indices like ENc (Effective Number ofCodon) and Fop (Frequency of optimized codons) werecalculated using DAMBE software [23]) and CodonW re-spectively [24]. GC3s values were calculated usingCodonO webserver [25]. Coding sequences of elongationfactors were retrieved from GeneDB. A comparison of theCAI values of the elongation factors with the CAI valuesof IPCS, SPT and SPL was made to determine the codonusage bias in each of the three genes.

Results and discussionPhylogenetic tree construction for the sphingolipidmetabolismPhylogenetic analysis provides a basis for understandingthe diversity within the conserved protein families. Phylo-genetic tree for the sphingolipid metabolism included atotal of 172 protein sequences belonging to the four pro-tein families. The optimal tree was obtained and the sumof branch length was 46.11 (Figure 2). The rate of aminoacid substitutions per site was 0.2. From the phylogenetictree, it was observed that IPCS and SPL protein familieswere phylogenetically distant from SPT protein family.IPCS in Leishmania showed resemblance to the IPCS pro-tein in plants as compared to other fungal groups.

probability along with functional divergence.


Functional divergencePhylogenetic analysis doesn’t provide much informa-tion for genes that have diverged by accumulating onlyfew mutations. For such genes, analysis of the Type Ifunctional divergence provides patterns of sequencevariations across the gene family. Site specific func-tional divergence of a gene across a set of homologousgene sequences can be analyzed using Type-I func-tional divergence. Functional divergence correlateswith the kind of selection pressure operating over theprotein (positive, neutral or purifying), defining the ex-tent of amino acid conservation. Evolutionary pressuresoperating over the parasitic proteins vary from oneamino acid to another. Amino acids that are of func-tional importance, are evolutionary conserved, on theother hand amino acids experiencing high rates ofsequence divergence alter the basic function of a pro-tein. The coefficient of functional divergence for the

Figure 4 Conservation profile of the protein families. A) IPCS protein familymarked in yellow indicate the presence of positive selection while those coloured

PAP2c (0.69) and SPL (0.36) protein families was muchhigher than that of SPT (0.36) protein family (Figure 3).Site specific posterior probability analysis indicatedthat PAP2c and SPL protein families diverge from theSPT family.

Specificity determining positions [SDP]Gene duplication, deletion events along with the evolutio-nary selection pressures alter the basic biochemicalproperties of a protein like ligand binding, protein-proteininteractions and the specificity towards the substrate. Theamino acid positions which vary only in certain subgroupsand alter the specificity of a protein are called the Specifi-city determining positions (SDP’s). Alignment positions,accounting for such functional specificity of the three en-zymes in L. major were mapped and analyzed for theirconservation. A total of 34 positions were identified asSDP’s in all the four groups (Additional file 1: Table S3).

B) SPL protein family C) SPT1 protein family D) SPT2 protein family. Residuesin gray or pink indicate neutral and purifying selection respectively.

Figure 5 ENc vs GC3s plot for A) IPCS protein family B) SPL protein family C) SPT1 protein family and D) SPT2 protein family. Red dotsrepresent the ENc and GC3s values of different Leishmania species. Lower ENc values together with the high GC3s value indicated the effect ofselection pressure over the parasitic proteins.

Table 1 Codon usage bias indices for IPCS, SPL and SPT genes

Enzyme name Species CAI ENc GC3S FOP Highest RSCU

IPCS L.major 0.604 50.34 0.72 0.362 UUG (Leu)

L.donovani 0.623 48.61 0.70 0.385 CGC (Arg)

L.braziliensis 0.573 48.21 0.69 0.394 CGC (Arg)

SPL L.major 0.681 44.22 0.78 0.39 CUG (Leu)

L.infactum 0.685 43.26 0.80 0.37 CGC (Arg)

SPT-1 L.major 0.694 46.45 0.77 0.323 CUG (Leu)

SPT-2 L.major 0.720 40.29 0.83 0.373 GUU (Val)

RSCU (Relative synonymous codon usage was calculated to estimate the number of cotton under codon usage bias. CUB indices calculate are a) CAI – Codonadaptability index b) ENc – Effective number of codons c) GC3S – GC content at third synonymous position d) FOP – Frequency of optimized codons. A comparisonof these indices between Leishmania and other homologous species is made. Range of indices between homologous species is given in the brackets. IPCS, SPT andSPL genes of Leishmania experience higher codon usage bias as compared to their homologues.


Table 2 CAI values of IPCS, SPL and SPT genes incomparison with CAI values of the highly expressedelongation factors of Leishmania

Enzyme Species CAI Elongation factor CAI

IPCS L.major 0.604 eEF1B beta 1 (LmjF.34.0820) 0.866

L.donovani 0.623

L.braziliensis 0.573

SPL L.major 0.681 EF2-1 (LmjF.36.0180) 0.821

L.infactum 0.685

SPT-1 L.major 0.694 EF1G (LmjF.09.0970) 0.847

SPT-2 L.major 0.720 EF-Tu (LmjF.18.0740) 0.768

EF1A (LmjF.17.0081) 0.880

SPT gene showed a higher CAI value as compared to IPCS and SPL genes.


Selection pressure over the protein familiesSelection pressure operating over protein families can beestimated using the ratio of non-synonymous substitutionsand the synonymous substitutions. The extent of positiveselection pressure, determines the relative rate of mutabilityof any amino acid (dN/dS ratio > 1). Purifying selection onthe other hand, promotes conservation of the amino acids(DN/DS ratio < 1). Sphingolipid metabolism of parasitesremains highly conserved among several parasites. Asevident from the CONSURF analysis, most of the aminoacids of all the three protein families fall under the ef-fect of purifying selection and are functionally con-served across closely related species (Additional file 1:Figure S1A-D, Figure 4A-D). Amino acids in the IPCSprotein family were less conserved as compared to SPL andSPT protein families. Two sites (HIS20 and CYS30) in theIPCS protein were under the influence of positive selection(Figure 4A). Relative mutability of such sites is much higherand hence inhibitor designing against the binding pocketscontaining these residues should be avoided in order to cutdown the probability of developing drug resistance.Studies of synonymous codon usage also reveal infor-

mation about the molecular evolution of individualgenes. A gene can be characterized not only by its aminoacid sequence but also by its codon usage shaped up bythe balance between mutational bias and natural selection.Differences in the codon bias between species arise as aconsequence of the selection pressure, which results inthe non-uniform usage of synonymous codons within agene. Bias can be either codon specific or gene specific.Optimal codons can also be defined by their nucleotidechemistry (GC content) and the codon usage bias. Genesdisplay a non-random usage of synonymous codons and ameasure of this non-randomness is RSCU (Relative syn-onymous codon usage). Codons with an RSCU valuehigher than 1 are used more frequently than expected,while codons with an RSCU value less than 1 indicate alesser preference of the particular codon [26]. To under-stand the codon usage pattern for IPCS, SPL and SPT

protein families, RSCU values were calculated and thecodon adaptability index (CAI) was obtained [27]. CAIvalues of IPCS gene were slightly lesser than that of SPTor SPL. At least one of the codons for each of the threegenes had a RSCU value greater than 1. We comparedthe ENc values of genes in Leishmania with that ofother homologues, ENc values of Leishmania specieswere lesser than other organisms. This indicates thepossibility of selection pressure, guiding the transla-tional selection of optimal codons in Leishmania [28]. Aplot of ENc vs GC3s indicated that the ENc values gen-erally showed negative correlation with the GC content.All the genes tested in Leishmania had a higher pre-dominance of G and C ending codons. Moreover GCcontent at the third synonymous position in Leishmaniaspecies was higher than that of other species. Codonusage pattern of all the genes therefore appears to bedetermined by the GC3s value (Figure 5). Higher GCcontent, lower Fop values along with lower ENc valuessuggest that there exists a moderate bias in the usage ofsynonymous codons in different Leishmania species.Codon usage bias was much more predominant in theSPT and SPL genes as compared to IPCS (Table 1).Elongation factors show higher expression and stron-

ger codon usage bias. A comparison of the CAI valuesof the various elongation factors of Leishmania withthe genes coding for our target proteins was done. TheCAI value of SPT gene was comparable with that ofCAI values of elongation factors. SPT gene thereforeshowed stronger codon usage bias than SPL or IPCS(Table 2). In due consideration of the role of selectionpressure deciding the evolvability of target proteins, itcan be concluded that the three target proteins weremoderately conserved and given their importance in thesphingolipid metabolism, these proteins could serve asgood drug targets.

ConclusionThe key enzymes (IPCS, SPL and SPT) of the sphingo-lipid metabolism of L.major were studied in relation toother members of the same protein family. The presentstudy supports the classification of sphingolipid metabo-lism with a high bootstrap value on the internal branches.To derive sufficient information about the kind of evo-lutionary pressure the three enzymes are being sub-jected to, functional divergence and conservation at thelevel of both amino acids and codons was studied. Func-tional divergence analysis indicated that PAP2c familydiverged from SPL and SPT. Amino acids accountingfor functional specificity (SDP’s) of the three enzymes inL.major were mapped. Along with the functional diver-gence, the selection pressures over the protein familieswere assessed. The effect of selection pressure was morepredominant at the codon level as indicated by the CUB


indices. IPCS gene showed a lower codon usage bias ascompared to SPL and SPT genes. At the protein level, theeffect of purifying selection was largely predominant andthis accounted for the functional conservation of the threedrug targets.

Additional file

Additional file 1: Supplementary file.

AbbreviationsER: Endoplasmic reticulum; IPCS: Inositol phosphoryl ceramide synthase;SPL: Sphingosine 1-P-lyase; SPT1: Serine palmitoyl transferase 1; SPT2: Serinepalmitoyl transferase 2; SMS: Sphingomyelin synthase; NJ: Neighbour-joining;ME: Minimum evolution; SDP: Specificity determining positions;RSCU: Relative synonymous codon usage; ENc: Effective number of codon;Fop: Frequency of optimized codons; CAI: Codon adaptation index;CDS: Coding sequence; MSA: Multiple sequence alignment; CUB: Codonusage bias.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionConceived and designed the experiments: SHS, SOS, VM. SOS and VMparticipated in the acquisition of sequences. SOS, VM participated in thephylogenetic tree construction and functional divergence analysis. VM SOScontributed in the selection pressure assessment of the protein families. SHSVM SOS contributed to the data analysis. SHS, VM, SOS wrote themanuscript. All the authors read and approve the final manuscript.

AcknowledgementVineetha Mandlik acknowledges the financial support as Senior Research Fellowof Department of Biotechnology, Government of India. The work was supportedby Department of Biotechnology, Government of India (BT/PR6037/GBD/27/372/2012). The authors would also like to thank Dr. Shekhar C. Mande for supportingthe Bioinformatics and High Performance Computing Facility at National Centrefor Cell Science, Pune, India.

Received: 13 December 2013 Accepted: 13 June 2014Published: 21 June 2014

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doi:10.1186/1471-2148-14-142Cite this article as: Mandlik et al.: Molecular evolution of the enzymesinvolved in the sphingolipid metabolism of Leishmania: selection pressurein relation to functional divergence and conservation. BMC EvolutionaryBiology 2014 14:142.

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